Transgenic animal models are essential experimental tools for unraveling gene function in normal development and disease. An ideal transgene recapitulates the endogenous gene expression pattern. To this end, it should encompass all the regulatory sequences required for recapitulating endogenous expression. Since these regulatory sequences are often far apart, such transgenic constructs frequently need to be a few hundred kilo bases (kb) of DNA in length. Large fragments (100-200 kb) of the genomes of most animal model systems have been subcloned into bacterial artificial chromosomes (BACs). Such BACs can be modified through recombineering (recombinant genetic engineering). However, techniques that facilitate efficient BAC transgenesis are lacking. The goal of this proposal is to develop a technique for efficient insertion of BAC constructs into the genome of the model organism zebrafish. Past efforts show that injection of naked BAC DNA or co-injection of Tol2 transposase with BAC DNA flanked by Tol2 sites into fertilized zebrafish eggs results in comparable transgenesis rates of 0.5-5%. Here, we propose to test a modified Tol2-mediated BAC transgenesis approach. Specifically, we intend to (1) develop universal tools for incorporating transposase sites into any BAC, (2) test the versatility of our modified Tol2-mediated BAC transgenesis approach for use with BACs harboring small and large DNA fragments and, as a proof of principle, generate Cre reporter lines.